Comprehensive transcriptome analysis of extracellular RNA (exRNA) purified from human biofluids is challenging because of the low RNA concentration and compromised RNA integrity. Here, we describe an optimized workflow to (1) isolate exRNA from different types of biofluids and (2) to prepare messenger RNA (mRNA)-enriched sequencing libraries using complementary hybridization probes. Importantly, the workflow includes 2 sets of synthetic spike-in RNA molecules as processing controls for RNA purification and sequencing library preparation and as an alternative data normalization strategy. For complete details on the use and execution of this protocol, please refer to Hulstaert et al. (2020).Super-resolution microscopy (SRM) has been widely adopted to probe molecular distribution at excitatory synapses. We present an SRM paradigm to evaluate the nanoscale organization heterogeneity between neuronal subcompartments. Using mouse hippocampal neurons, we describe the identification of the morphological characteristics of nanodomains within functional zones of a single excitatory synapse. This information can be used to correlate structure and function at molecular resolution in single synapses. The protocol can be applied to immunocytochemical/histochemical samples across different imaging paradigms. For complete details on the use and execution of this protocol, please refer to Kedia et al. (2021).Here, we present a comprehensive protocol to analyze the roles of disease-related genes in synaptic transmission. We have developed a pipeline of electrophysiological techniques and combined these with optogenetics in the medial prefrontal cortex of mice. This methodology provides a cost-effective, faster, and easier screening approach to elucidate functional aspects of single genes in several regions in the mouse brain such as a specific layer of the mPFC. For complete details on the use and execution of this protocol, please refer to Nagahama et al. (2020) and Sacai et al. (2020).The 4,5-dimethoxy-2-nitrobenzyl (DMNB) photocaging group introduced into small biomolecules, peptides, oligonucleotides, and proteins is commonly used for spatiotemporal control of chemical and biological processes. Here, we describe the use of a DMNB-selective monoclonal antibody for non-covalent capture of chemically or biosynthetically produced proteins containing surface-exposed DMNB caging groups followed by light-controlled traceless decaging and release of the bound proteins into solution for a variety of downstream applications. For complete details on the use and execution of this protocol, please refer to Rakauskaitė et al. (2020).This protocol describes how to visualize surface protein-protein co-localization across a cell-cell interface between antigen-presenting γδ-T cells and CD4 T cells. By consolidating immunofluorescence assay, confocal microscopy and 3D imaging analysis, it enables assessment of interaction between cell surface proteins such as Δ42PD1 and TLR4 between co-cultured γδ-T and CD4 T cells. This protocol can be applied to study a surface protein of interest and its potential interaction with a target cell/protein at the cell-cell interface. For complete details on the use and execution of this profile, please refer to Mo et al. (2020).It remains challenging to generate reproducible, high-quality cDNA libraries from RNA derived from rare cell populations. Here, we describe a protocol for high-throughput RNA-seq library preparation, including isolation of 200 skeletal muscle stem cells from mouse tibialis anterior muscle by fluorescence-activated cell sorting and cDNA preparation. We also describe RNA extraction and cDNA preparation from differentiating mouse embryonic stem cells. For complete details on the use and execution of this protocol, please refer to Juan et al. https://www.selleckchem.com/products/atuveciclib-bay-1143572.html (2016) and Garcia-Prat et al. (2016).The quality and safety of food is an important issue to the whole society, since it is at the basis of human health, social development and stability. Ensuring food quality and safety is a complex process, and all stages of food processing must be considered, from cultivating, harvesting and storage to preparation and consumption. However, these processes are often labour-intensive. Nowadays, the development of machine vision can greatly assist researchers and industries in improving the efficiency of food processing. As a result, machine vision has been widely used in all aspects of food processing. At the same time, image processing is an important component of machine vision. Image processing can take advantage of machine learning and deep learning models to effectively identify the type and quality of food. Subsequently, follow-up design in the machine vision system can address tasks such as food grading, detecting locations of defective spots or foreign objects, and removing impurities. In this paper, we provide an overview on the traditional machine learning and deep learning methods, as well as the machine vision techniques that can be applied to the field of food processing. We present the current approaches and challenges, and the future trends.Characterising key components within functional ingredients as well as assessing efficacy and bioavailability is an important step in validating nutritional interventions. Machine learning can assess large and complex data sets, such as proteomic data from plants sources, and so offers a prime opportunity to predict key bioactive components within a larger matrix. Using machine learning, we identified two potentially bioactive peptides within a Vicia faba derived hydrolysate, NPN_1, an ingredient which was previously identified for preventing muscle loss in a murine disuse model. We investigated the predicted efficacy of these peptides in vitro and observed that HLPSYSPSPQ and TIKIPAGT were capable of increasing protein synthesis and reducing TNF-α secretion, respectively. Following confirmation of efficacy, we assessed bioavailability and stability of these predicted peptides and found that as part of NPN_1, both HLPSYSPSPQ and TIKIPAGT survived upper gut digestion, were transported across the intestinal barrier and exhibited notable stability in human plasma.